The principle of AIR is described in co-pending U.S. patent application Ser. No. 10/282,274, filed Oct. 28, 2002, now U.S. Pat. No. 7,292,349, issued Nov. 6, 2007. AIR exploits interference between reflections from the medium/coating and coating/substrate interfaces, exhibiting changes in reflectivity upon binding of biomolecules to the coating. In practice, using a silicon wafer having an oxide coating, judicious choice of incident angle and wavelength can be used with s-polarized light to obtain near complete destructive interference (i.e., reflectivity that is preferably less than about 10−5 or 10−6 under some circumstances) in the absence of a target molecule. The condition of near complete (or near perfect) destructive interference is removed upon target molecule binding. Thus, highly sensitive detection of even small quantities of a target molecule is possible.
While AIR using s-polarized light has proven to be a highly sensitive, simple analytical method for the quantitative detection of a variety of biomolecular analytes, it is much more easily carried out in a dry state, that is, with an air/oxide interface rather than with an aqueous/oxide interface. This is a consequence of the difference in the refractive indexes of the media (water≈1.33; air≈1), and the difference in incidence angles that are required to achieve the condition of near complete destructive interference in these media. Using silicon/oxide as the substrate/coating system, in air this angle is about 70.5 degrees whereas in water this angle is about 85.5. The system that has proven to be optimal in air, however, could not be optimized for use in aqueous environments.
There is a need for obtaining an AIR system that is operable in aqueous environments yet maintains the sensitivity of the AIR system that is optimal for “dry” state described above. Detection in aqueous environments is preferred for most biological targets, because the aqueous condition more accurately replicates that condition under which the target molecule normally exists. This is also believed to improve protein viability, as well as allow for different types of measurements such as the acquisition of kinetic information (e.g., on/off rates for coupling to a probe, comparative affinity between two different target molecules, etc.) and even continuous flow for real-time sensors. There exists a substantial need, therefore, for an improved AIR system that is capable of achieving these results.
The present invention is directed to overcoming these and other deficiencies in the art.